Artemisinin-based drugs are the most effective medicine against multidrug-resistant Plasmodium spp., the parasite that causes malaria. To this day, wormwood A. annua L. is the sole commercial source of artemisinin, where it is produced in minor amounts. The artemisinin yield depends on numerous poorly regulated agricultural factors and the genetic variability of this non-domesticated plant. This has aroused significant interest in the development of heterologous expression platforms for artemisinin production. Previously, we obtained lines of Chrysanthemum morifolium Ramat. (C. morifolium Ramat.), cvs. White Snowdon and Egyptianka, transformed with artemisinin biosynthesis genes. Here, we report the results of an analysis of artemisinin production in transgenic chrysanthemums. Transcription of heterologous amorpha-4,11-diene monooxygenase and cytochrome P450 reductase genes in transgenic lines was confirmed using high-resolution melting analysis. Artemisinin accumulation was detected using GC-MS in White Snowdon plants, but not in Egyptianka ones, thereby demonstrating the possibility of transplanting active artemisinin biosynthetic pathway into chrysanthemum. Ways of increasing its content in producer plants are discussed.

Floral guides are patterned cues that direct the pollinator to the plant reproductive organs. The spatial distribution of showy visual and olfactory traits allows efficient plant-pollinator interactions. Data on the mechanisms underlying floral volatile patterns or their interactions with pollinators are lacking. Here we characterize the spatial emission patterns of volatiles from the corolla of the model plant Petunia x hybrida and reveal the ability of honeybees to distinguish these patterns. Along the adaxial epidermis, in correlation with cell density, the petal base adjacent to reproductive organs emitted significantly higher levels of volatiles than the distal petal rim. Volatile emission could also be differentiated between the two epidermal surfaces: emission from the adaxial side was significantly higher than that from the abaxial side. Similar emission patterns were also observed in other petunias, Dianthus caryophyllus (carnation) and Argyranthemum frutescens (Marguerite daisy). Analyses of transcripts involved in volatile production/emission revealed lower levels of the plasma-membrane transporter ABCG1 in the abaxial versus adaxial epidermis. Transient overexpression of ABCG1 enhanced emission from the abaxial epidermis to the level of the adaxial epidermis, suggesting its involvement in spatial emission patterns in the epidermal layers. Proboscis extension response experiments showed that differences in emission levels along the adaxial epidermis, that is, petal base versus rim, detected by GC-MS are also discernible by honeybees.

The genetic code, once believed to be universal and immutable, is now known to contain many variations and is not quite universal. The basis for genome recoding strategy is genetic code variation that can be harnessed to improve cellular properties. Thus, genome recoding is a promising strategy for the enhancement of genome flexibility, allowing for novel functions that are not commonly documented in the organism in its natural environment. Here, the basic concept of genetic code and associated mechanisms for the generation of genetic codon variants, including biased codon usage, codon reassignment, and ambiguous decoding, are extensively discussed. Knowledge of the concept of natural genetic code expansion is also detailed. The generation of recoded organisms and associated mechanisms with basic targeting components, including aminoacyl-tRNA synthetase–tRNA pairs, elongation factor EF-Tu and ribosomes, are highlighted for a comprehensive understanding of this concept. The research associated with the generation of diverse recoded organisms is also discussed. The success of genome recoding in diverse multicellular organisms offers a platform for expanding protein chemistry at the biochemical level with non-canonical amino acids, genetically isolating the synthetic organisms from the natural ones, and fighting viruses, including SARS-CoV2, through the creation of attenuated viruses. In conclusion, genome recoding can offer diverse applications for improving cellular properties in the genome-recoded organisms.

The papain-like cysteine proteases (PLCPs) are the most abundant family of cysteine proteases in plants, with essential roles in biotic/abiotic stress responses, growth and senescence. Papain, bromelain and ficin are widely used in food, medicine and other industries. In this study, 31 PLCP genes (FcPCLPs) were identified in the fig (Ficus carica L.) genome by HMM search and manual screening, and assigned to one of nine subfamilies based on gene structure and conserved motifs. SAG12 and RD21 were the largest subfamilies with 10 and 7 members, respectively. The FcPCLPs ranged from 1,128 to 5,075 bp in length, containing 1–10 introns, and the coding sequence ranged from 624 to 1,518 bp, encoding 207–505 amino acids. Subcellular localization analysis indicated that 24, 2, and 5 PLCP proteins were targeted to the lysosome/vacuole, cytoplasm and extracellular matrix, respectively. Promoter (2,000 bp upstream) analysis of FcPLCPs revealed a high number of plant hormone and low temperature response elements. RNA-seq revealed differential expression of 17 FcPLCPs in the inflorescence and receptacle, and RD21 subfamily members were the major PLCPs expressed in the fruit; 16 and 5 FcPLCPs responded significantly to ethylene and light, respectively. Proteome analyses revealed 18 and 5 PLCPs in the fruit cell soluble proteome and fruit latex, respectively. Ficins were the major PLCP in fig fruit, with decreased abundance in inflorescences, but increased abundance in receptacles of commercial-ripe fruit. FcRD21B/C and FcALP1 were aligned as the genes encoding the main ficin isoforms. Our study provides valuable multi-omics information on the FcPLCP family and lays the foundation for further functional studies.

Abstract BACKGROUND Thaumatin II, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), shows promise as a zero-calorie sweetener for use in the food and pharmaceutical industries and for improving the taste of fruit. RESULTS We report on the stability of thaumatin in salted and pickled tomatoes, as well as on the effect of thaumatin on the taste quality of processed tomatoes. Fruit of tomato cv. Yalf, transformed with the thaumatin II gene were salted and pickled and then stored for 6 months. Western blot analysis showed relative thaumatin II stability at salting; its content in processed fruits was 62?83% of the initial level depending in the studied line. In pickled tomatoes, thaumatin II content was decreased by up to 25% of the initial amount. Both salted and pickled tomatoes had a sweet taste with a typical thaumatin aftertaste. Salted tomatoes were characterized as being sweeter than pickled tomatoes. The overall taste of pickled tomatoes was rated by panellists as significantly better compared to that of salted or non-processed ones. CONCLUSION In the present study, we have shown that tomatoes expressing supersweet protein thaumatin II can be used for processing under mild conditions, including salting and pickling. ? 2021 Society of Chemical Industry.

Artemisinin-based drugs are the most effective medicine for the malaria treatment. To date, the main method of artemisinin production is its extraction from wormwood plants Artemisia annua L. Due to the limitation of this source, considerable efforts are now directed to the development of methods for artemisinin production using heterologous expression systems. Artemisinin is a sesquiterpene lactone, synthesized through the cyclization of farnesyl diphosphate involved in other sesquiterpene biosynthetic systems. Chrysanthemum species as well as A. annua, belong to Asteraceae family, and had been characterized by containing highly content of sesquiterpenes and their precursors. This makes chrysanthemum a promising target for the production of artemisinin in heterologous host plants. Chrysanthemum (C. morifolium Ramat.) was transformed by Agrobacterium tumefaciens carrying with the binary vectors p1240 and p1250, bearing artemisinin biosynthesis genes coding: amorpha-4,11-diene synthase, artemisinic aldehyde D11(13) reductase, amorpha-4,11-diene monooxygenase (p1240 was targeted to the mitochondria and p1250 was targeted to the cytosol), cytochrome P450 reductase from A. annua, as well as yeast truncated 3-hydroxy-3-methylglutarylcoenzyme A reductase. This study obtained 8 kanamycin-resistant lines after transformation with the p1240 and 2 lines from p1250. All target genes were detected in 2 and 1 transgenic lines of the 2 vectors. The transformation frequency of all target genes were 0.33% and 0.17% for p1240 and p1250, relative to the total transformed explant numbers. RT-PCR analysis revealed the transcription of all transferred genes in two lines obtained after transformation with the p1240 vector, confirming the possibility of transferring genetic modules encoding entire biochemical pathways into the chrysanthemum genome. This holds promise for the development of a chrysanthemum-based expression system to produce non-protein substances, such as artemisinin.

Summary The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spillover infection in December 2019 has caused an unprecedented pandemic. SARS-CoV-2, as other coronaviruses, binds its target cells through the angiotensin-converting enzyme 2 (ACE2) receptor. Accordingly, this makes ACE2 research essential for understanding the zoonotic nature of coronaviruses and identifying novel drugs. Here we present a systematic analysis of the ACE2 conservation and co-evolution protein network across 1,671 eukaryotes, revealing an unexpected conservation pattern in specific metazoans, plants, fungi, and protists. We identified the co-evolved protein network and pinpointed a list of drugs that target this network by using data integration from different sources. Our computational analysis found widely used drugs such as nonsteroidal anti-inflammatory drugs and vasodilators. These drugs are expected to perturb the ACE2 network affecting infectivity as well as the pathophysiology of the disease.

Floral pigmentation is of major importance to the ornamental industry, which is constantly searching for cultivars with novel colors. Goldenrod (Solidago canadensis) has monochromatic yellow carotenoid-containing flowers that cannot be modified using classical breeding approaches due to a limited gene pool. To generate Solidago with novel colors through metabolic engineering, we first developed a procedure for its regeneration and transformation. Applicability of different cytokinins for adventitious regeneration was examined in the commercial cv. Tara, with zeatin yielding higher efficiency than 6-benzylaminopurine or thidiazuron. A comparison of regeneration of commercial cvs. Tara, Golden Glory and Ivory Glory revealed Tara to be the most potent, with an efficiency of 86% (number of shoots per 100 leaf explants). Agrobacterium-based transformation efficiency was highest for cv. Golden Glory (5 independent transgenic shoots per 100 explants) based on kanamycin selection and the GUS reporter gene. In an attempt to promote anthocyanin biosynthesis, we generated transgenic Solidago expressing snapdragon (Antirrhinum majus) Rosea1 and Delila, as well as Arabidopsis thaliana PRODUCTION OF ANTHOCYANIN PIGMENT 1 (PAP1) transcription factors. Transgenic cv. Golden Glory expressing cauliflower mosaic virus 35S-driven PAP1 generated red flowers that accumulated delphinidin and its methylated derivatives, as compared to control yellow flowers in the GUS-expressing plants. The protocol described here allows efficient engineering of Solidago for novel coloration and improved agricultural traits. © Copyright © 2019 Skaliter, Ravid, Shklarman, Ketrarou, Shpayer, Ben Ari, Dvir, Farhi, Yue and Vainstein.

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including a reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer C. orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found outside Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent. This article is protected by copyright. All rights reserved.

BACKGROUND: Fig fruit are highly perishable at the tree-ripe (TR) stage. Commercial-ripe (CR) fruit, which are harvested before the TR stage for their postharvest transportability and shelf-life advantage, are inferior to TR fruit in size, color and sugar content. The succulent urn-shaped receptacle, serving as the protective structure and edible part of the fruit, determines fruit quality. Quantitative iTRAQ and RNA-Seq were performed to reveal the differential proteomic and transcriptomic traits of the receptacle at the two harvest stages. RESULTS: We identified 1226 proteins, of which 84 differentially abundant proteins (DAPs) were recruited by criteria of abundance fold-change (FC) ≥1.3 and p < 0.05 in the TR/CR receptacle proteomic analysis. In addition, 2087 differentially expressed genes (DEGs) were screened by ≥2-fold expression change: 1274 were upregulated and 813 were downregulated in the TR vs. CR transcriptomic analysis. Ficin was the most abundant soluble protein in the fig receptacle. Sucrose synthase, sucrose-phosphate synthase and hexokinase were all actively upregulated at both the protein and transcriptional levels. Endoglucanase, expansin, beta-galactosidase, pectin esterase and aquaporins were upregulated from the CR to TR stage at the protein level. In hormonal synthesis and signaling pathways, high protein and transcriptional levels of aminocyclopropane-1-carboxylate oxidase were identified, together with a few diversely expressed ethylene-response factors, indicating the potential leading role of ethylene in the ripening process of fig receptacle, which has been recently reported as a non-climacteric tissue. CONCLUSIONS: We present the first delineation of intra- and inter-omic changes in the expression of specific proteins and genes of TR vs. CR fig receptacle, providing valuable candidates for further study of fruit-quality formation control and fig cultivar innovation to accommodate market demand.

Plant vacuoles are the largest compartment in plant cells, occupying more than 80% of the cell volume. A variety of proteins, sugars, pigments and other metabolites are stored in these organelles (Paris et al., 1996; Olbrich et al., 2007). Flowers produce a variety of specialized metabolites, some of which are unique to this organ, such as components of pollination syndromes, i.e., scent volatiles and flavonoids (Hoballah et al., 2007; Cna'ani et al., 2015). To study the compounds stored in floral vacuoles, this compartment must be separated from the rest of the cell. To enable isolation of vacuoles, protoplasts were first generated by incubating pierced corollas with cellulase and macrozyme enzymes. After filtering and several centrifugation steps, protoplasts were separated from the debris and damaged/burst protoplasts, as revealed by microscopic observation. Concentrated protoplasts were lysed, and vacuoles were extracted by Ficoll-gradient centrifugation. Vacuoles were used for quantitative GC-MS analyses of sequestered metabolites. This method allowed us to identify vacuoles as the subcellular accumulation site of glycosylated volatile phenylpropanoids and to hypothesize that conjugated scent compounds are sequestered in the vacuoles en route to the headspace (Cna'ani et al., 2017).

The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

Summary Emission of volatiles at advanced stages of flower development is a strategy used by plants to lure pollinators to the flower. We reveal that GA negatively regulates floral scent production in petunia. We used Agrobacterium-mediated transient expression of GA-20ox in petunia flowers and a virus-induced gene silencing approach to knock down DELLA expression, measured volatile emission, internal pool sizes and GA levels by GC-MS or LC–MS/MS, and analyzed transcript levels of scent-related phenylpropanoid-pathway genes. We show that GA has a negative effect on the concentrations of accumulated and emitted phenylpropanoid volatiles in petunia flowers; this effect is exerted through transcriptional/post-transcriptional downregulation of regulatory and biosynthetic scent-related genes. Both overexpression of GA20-ox, a GA-biosynthesis gene, and suppression of DELLA, a repressor of GA-signal transduction, corroborated GA's negative regulation of floral scent. We present a model in which GA-dependent timing of the sequential activation of different branches of the phenylpropanoid pathway during flower development may represent a link between the showy traits controlling pollinator attraction, namely color and scent.

Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography–mass spectrometry (GC–MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger β-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC–MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites.

Combined metabolomic and transcriptomic analyses were carried out with fig cultivar Green Peel and its color mutant "Purple Peel." Five and twenty-two metabolites were identified as having significantly different contents between fruit peels of the two cultivars at young and mature stages, respectively. Cyanidin O-malonylhexoside demonstrated a 3,992-fold increase in the mature purple peel, the first identification of a major cyanidin in fig fruit; cyanidin 3-O-glucoside, cyanidin O-malonylhexoside O-hexoside and cyanidin-3,5-O-diglucoside were upregulated 100-fold, revealing the anthocyanins underlying the purple mutation. Beyond the visible differences, there was very significant accumulation of the colorless flavonoids procyanidin B1, luteolin-3',7-di-O-glucoside, epicatechin and quercetin-3-O-rhamnoside in the mature "Purple Peel" compared to "Green Peel." At the young stage, only cyanidin O-malonylhexoside, cyanidin O-malonylhexoside O-hexoside and esculetin were upregulated a few fold in the mutant. Transcriptome analysis revealed a downregulated expression trend of genes encoding phenylpropanoid and flavonoid biosynthetic pathway enzyme in the young "Purple Peel" compared to the young "Green Peel," whereas significant and simultaneous upregulation was revealed in almost all of the flavonoid and anthocyanin pathway components and relevant transcription factors in the mature-stage mutant. The role of R2R3-MYB transcription factors in the color morph mutation and its possible relation to the activity of retrotransposons are discussed. Moreover, large-scale upregulation of small heat-shock protein genes was found in the mature mutant. This is the first work to reveal comprehensive metabolome and transcriptome network changes underlying a fig mutation in a single horticultural attribute, and its profound effects on fruit nutrition and quality.

Thaumatin, a supersweet protein from the African plant katemfe (Thaumatococcus daniellii Benth.), is a promising zero-calorie sweetener for use in the food and pharmaceutical industries. Due to limited natural sources of thaumatin, its production using transgenic plants is an advantageous alternative. We report a simple protocol for purification of recombinant thaumatin II from transgenic tomato. Thaumatin was extracted from ripe tomato fruit in a low-salt buffer and purified on an SP-Sephacryl column. Recombinant thaumatin yield averaged 50 mg/kg fresh fruit. MALDI-MS analysis showed correct processing of thaumatin in tomato plants. The recombinant thaumatin was indistinguishable from the native protein in a taste test. The purified tomato-derived thaumatin had an intrinsic sweetness with a threshold value in taste tests of around 50 nM. These results demonstrate the potential of an expression system based on transgenic tomato plants for production of recombinant thaumatin for the food and pharmaceutical industries.

Increasing temperatures due to changing global climate are interfering with plant-pollinator mutualism, an interaction facilitated mainly by floral colour and scent. Gas chromatography-mass spectroscopy analyses revealed that increasing ambient temperature leads to a decrease in phenylpropanoid-based floral scent production in two Petunia × hybrida varieties, P720 and Blue Spark, acclimated at 22/16 or 28/22 °C (day/night). This decrease could be attributed to down-regulation of scent-related structural gene expression from both phenylpropanoid and shikimate pathways, and up-regulation of a negative regulator of scent production, emission of benzenoids V (EOBV). To test whether the negative effect of increased temperature on scent production can be reduced in flowers with enhanced metabolic flow in the phenylpropanoid pathway, we analysed floral volatile production by transgenic 'Blue Spark' plants overexpressing CaMV 35S-driven Arabidopsis thaliana production of anthocyanin pigments 1 (PAP1) under elevated versus standard temperature conditions. Flowers of 35S:PAP1 transgenic plants produced the same or even higher levels of volatiles when exposed to a long-term high-temperature regime. This phenotype was also evident when analysing relevant gene expression as inferred from sequencing the transcriptome of 35S:PAP1 transgenic flowers under the two temperature regimes. Thus, up-regulation of transcription might negate the adverse effects of temperature on scent production.

The mechanism underlying the emission of phenylpropanoid volatiles is poorly understood. Here, we reveal the involvement of PH4, a petunia MYB-R2R3 transcription factor previously studied for its role in vacuolar acidification, in floral volatile emission. We used the virus-induced gene silencing (VIGS) approach to knock down PH4 expression in petunia, measured volatile emission and internal pool sizes by GC-MS, and analyzed transcript abundances of scent-related phenylpropanoid genes in flowers. Silencing of PH4 resulted in a marked decrease in floral phenylpropanoid volatile emission, with a concurrent increase in internal pool levels. Expression of scent-related phenylpropanoid genes was not affected. To identify putative scent-related targets of PH4, we silenced PH5, a tonoplast-localized H(+) -ATPase that maintains vacuolar pH homeostasis. Suppression of PH5 did not yield the reduced-emission phenotype, suggesting that PH4 does not operate in the context of floral scent through regulation of vacuolar pH. We conclude that PH4 is a key floral regulator that integrates volatile production and emission processes and interconnects two essential floral traits - color and scent.